Our work has been the first to demonstrate the brain's intrinsic capacity to regulate expression of hemostasis factors. Given the overwhelming importance of thromboembolic occlusion in stroke, defining molecular mechanisms of brain-specific hemostasis is a critical issue. Recent advances in genetics of ischemic stroke have emphasized the role of the phospodiesterase-4 (PDE4) enzymatic pathway. Initial work has demonstrated that low expression of PDE4D isoforms are associated with increased risk for both carotid and cardiogenic stroke. For the current project, we will focus on regulation of brain fibrinolysis via the PDE4 pathway, and its relationship with ischemic stroke. Our preliminary data show a) substantial linkage between PDE4D and tissue plasminogen activator expression, and b) increased infarct size with PDE4 inhibition. We have previously demonstrated an important role for brain pericytes in regulating expression of tPA and the serpin protease nexin-1 (PN-1). We propose to define the role of PDE4 as regulator and mediator of pericye and astrocyte effects on endothelial fibrinolysis. We will incorporate an in vitro model of ischemia, oxygen-glucose deprivation, to fully analyze these effects. Moreover, given the increasing recognition of inflammation in pathogenesis of ischemic stroke, we will study endotoxin effects on brain microvascular fibrinolysis. Specifically, we will define how PDE4 may mediate pericyte-dependent brain microvascular endothelial hyper-responsiveness to endotxoin. We will also analyze the in vivo role of PDE4 and PDE4D in the brain microvascular response to endotoxin, using rats pretreated with rolipram and mice null for PDE4D. We will define the role of PDE4 as regulator of both blood-brain barrier formation as well as brain microvascular fibrinolysis, conducting both in vitro and in vivo studies. Finally, we will analyze the role of PDE4 and PDE4D expression in mouse and rat experimental stroke models, and define the role of fibrinolysis as mediator between PDE4/PDE4D expression and infarct size in experimental stroke models. PDE4 thus represents a potentially critical enzymatic link between fibrinolysis, the blood-brain barrier, and ischemic stroke. We are also prepared to investigate, as necessary, other cAMP-mediated pathways, and non-cAMP-mediated pathways, regulating fibrinolysis, inflammation, and blood brain barrier formation. Completion of these studies will provide new insights for the molecular mechanisms regulating brain microvascular fibrinolysis, allowing for development of new strategies to prevent and treat stroke by modification of brain microvascular hemostasis function.